For centuries, starter cultures of lactic acid bacteria have been used in food production due to their ability to convert sugars by fermentation into organic acids, predominantly lactic acid, and various metabolites associated with the development in the fermented food products of a desirable taste and flavour. Several lactic acid bacteria inherently produce hydrolytic enzymes including peptidases, proteases and lipolytic enzymes, the production of which may e.g. contribute to a desired flavour development in cheeses.
However, for industrial production of a wide range of fermented food products such as all the well-known traditional dairy products including yoghurt, acidophilus milk, butter and cheeses; fermented vegetables; fermented meat products and animal feed, a large range of lactic acid bacterial starter cultures, each being adapted to particular types of food products, are required. Such cultures are presently being selected from naturally occurring strains of lactic acid bacteria on the basis of characteristics such as their ability to ferment sugars present in the food product to be fermented, specific growth temperature requirements, production of desired flavouring compounds, the specific combination of which characteristics renders a specifically selected wildtype culture useful for the production of a particular food product but normally less useful for the production of others.
Obviously, this presently used procedure for developing useful lactic acid bacterial cultures by selection of naturally occurring strains is cumbersome and costly. Furthermore, it has proven difficult to provide starter culture strains which combine all of the required characteristics at an optimal level. Presently, this problem is usually solved by the use of starter cultures comprising a multiplicity of selected lactic acid bacterial strains each having one or several of the characteristics desirable for a particular food product. The necessity to use such mixed cultures will of course add to the costs in the manufacture of lactic acid bacterial starter cultures.
Based on their traditional and long term application in food manufacturing and the fact that they are considered as nonpathogenic, the lactic acid bacteria are generally recognized as safe (GRAS) food ingredients, even if they are present in a fermented food product as live bacteria at a very high number, such as 10.sup.8 to 10.sup.9 per g.
Currently, it is widely recognized that a substantial industrial need exists to find economically and technically more feasible ways of developing improved lactic acid bacteria for use as food or feed starter cultures or for the production of desired gene products including providing lactic acid bacteria which are useful for a wide range of applications. It is evident that recombinant DNA technology may provide the means to meet this need. In this context, it is crucial that lactic acid bacteria for food manufacturing which are developed by introduction of desired genes by use of gene technology can still be recognized as safe for consumption. It is therefore considered by the food industry that it is essential that recombinant lactic acid bacteria essentially contain only DNA of lactic acid bacterial origin including DNA from wildtype extrachromosomal plasmids frequently found in starter culture strains or non-lactic acid bacterial DNA which does not confer to the recombinant strains any hazardous phenotypic traits.
There have been several attempts of providing genetically improved lactic acid bacteria. Most of these attempts have been directed to the construction of recombinant expression vectors coding for desired gene products and capable of replicating in lactic acid bacteria. However, very few of these attempts have resulted in vectors comprising only lactic acid bacterial DNA.
In addition to their use as food starter cultures, lactic acid bacteria can be used as production strains for the manufacturing of desired biologically functional gene products such as pharmaceutically and immunologically active compounds.
As mentioned above, the present invention provides novel lactic acid bacterial regulatable gene expression systems which are based on modification of naturally occurring regulatory sequences which are operably associated or linked with a gene, whereby the expression of the gene can be altered significantly.
Inducible or regulatable gene expression systems are highly important for expression of genes encoding proteins that are either (i) toxic to the host organism, (ii) needed in large quantities, (iii) used to study the effect of particular gene functions on cellular metabolism or regulation or (iv) produced at a particular point in time or under particular environmental conditions. Whereas inducible expression systems have been developed for use in E. coli, only a few inducible expression systems for use in lactic acid bacteria have been described.
An example of a lactic acid bacterial inducible expression system is a system based on the lac promoter transcribing the lac genes of Lactococcus lactis. The lac promoter can be repressed by the LacR repressor and a six-fold induction of transcription can be obtained by replacing glucose in the growth medium with lactose (van Rooijen et al., 1992). This naturally occurring expression system has been combined with the T7 RNA polymerase/T7 promoter system from E. coli (Wells et al., 1993a,b; Steidler et al., 1995). The lac promoter controls the expression of T7 RNA polymerase, which recognizes the T7 promoter, allowing inducible expression of genes cloned downstream of the T7 promoter. This system has been used to produce tetanus toxin fragment C and murine interleukin-2.
Another example is the use of the dnaJ promoter transcribing the dnaJ gene of L. lactis, which has been used to generate inducible expression of a heterologous protein after heat shock induction (van Asseldonk et al., 1993). Increasing the temperature from 30.degree. C. to 42.degree. C. resulted in about four-fold induction of gene transcription.
Other examples of lactic acid bacterial inducible expression systems include the use of phage specific expression signals from lytic bacteriophages of L. lactis which can be applied to express heterologous genes upon phage infection (O'Sullivan et al., 1996), and systems based on induction of gene expression by supplementing the bacterial growth medium with an inducer substance such as the toxic antitumour antibiotic Mitomycin C (Nauta et al., 1996) or a bacteriocin such as nisin (Ruyter et al., 1996, EP 0712 935 A2).
Accordingly, all the known methods of controlling gene expression in lactic acid bacteria are based upon the addition to the growth medium of inducing compounds or bacteriophages, or on temperature shifts, i.e. exogenously added factors which may not be acceptable according to regulatory safety requirements or which are not economically or industrially feasible in the context of industrial use of lactic acid bacteria in food or feed manufacturing or in the manufacturing of desired gene products.
It has recently been discovered that it is possible to isolate lactic acid bacterial promoters which are inducible or regulatable by the presence/absence or the concentration of one or more environmental factors associated with conventional lactic acid bacterial industrial production methods such as pH, growth temperature, composition of the growth medium including the ionic strength/NaCl content, the presence/absence of purine nucleotide precursors and/or the growth phase/growth rate of the bacterium (WO 94/16086, Israelsen et al., 1995).
It is evident that regulatable expression systems based on such environmental or growth condition factors as mentioned above, and which are normally present in industrial culture media for lactic acid bacteria either initially or during the culturing, will represent a highly attractive approach for regulating the production of homologous or heterologous gene products in lactic acid bacteria. However, in order for the application of these regulatable expression systems to be successful, the selected promoter must be effective and lead to the production of a desired protein in sufficiently high amounts under industrial conditions to facilitate an economically viable production or manufacturing process. However, it has been found that such otherwise useful naturally occurring regulatable lactic acid bacterial promoters may only have a relatively weak promoter activity.
It has now been discovered that the activity of such naturally occurring inducible or regulatable lactic acid bacterial promoters can be increased by modifying the nucleotide region in which the promoter is located and, most importantly, that such an increased promoter activity can be obtained without reducing or eliminating the inducibility by the above mentioned growth condition factors. The invention has also made it possible to provide sets or panels comprising lactic acid bacteria producing a desired gene product at different levels under the same conditions. Additionally, it has also been found that the modification of the promoter region sequences may result in strains having a modulated expression level under induced conditions as compared to the regulation by the corresponding non-modified promoter region.